Analysis of site-directed mutations in the alpha- and beta-subunits of Klebsiella pneumoniae nitrogenase.
Using directed mutagenesis, amino acid substitutions have been made in the alpha- and beta-subunits of the klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserved cysteine or tyrosine residues. Nif+, Nif- and intermediate phenotypes have been obtained. To extend our earlier biochemical characterization (Kent et al., 1989) the electrophoretic mobility of component 1 of the mutant and wild-type nitrogenases has been analysed by non-denaturing gel electrophoresis. The major and minor forms of component 1 separated by this methodology have been probed for by using both polyclonal and monoclonal antibodies. All Nif+ mutants exhibited a distribution of electrophoretic forms of component 1 comparable to the wild type, and the abundance of the major form found in purified nitrogenase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif- mutants exhibited either a major low-mobility form or a fast-moving form. Analysis of nitrogenase polypeptides synthesized in the absence of co-factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the alpha-subunit produces component 1 defective in its interaction with FeMoco. Substitution of other conserved cysteine residues by alanine appears to prevent early steps in nitrogenase assembly or to promote degradation. Two single mutations (cysteine 89 to alanine in the alpha-subunit and cysteine 94 to alanine in the beta-subunit) which are tightly Nif- can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.
Kent
H M
HM
AFRC Institute of Plant Science Research, University of Sussex, Brighton, UK.
Baines
M
M
Gormal
C
C
Smith
B E
BE
Buck
M
M
D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics*; D008983:Molybdoferredoxin / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype
D008983:Molybdoferredoxin; D009591:Nitrogenase